Is silver diammine fluoride effective in arresting dental caries in cavitated primary molars?

CLINICAL QUESTION: To evaluate the efficacy of silver diamine fluoride (SDF) in arresting dental caries in cavitated caries lesions in primary molars. DATA SOURCES: A systematic search was carried out in PubMed, Scopus, and Embase. Furthermore, cross-referencing was performed using the references lists of full-text articles and grey literature was also retrieved for eligible studies. Two independent reviewers were responsible for study selection and data extraction. STUDY SELECTION: Randomized and non-randomized clinical studies that evaluated the caries arrest rate of SDF compared to no treatment or any other type of non-invasive or minimally-invasive treatment were included. Only publications in the English, Italian and French language and with a minimum follow-up of 6 months were considered for study eligibility. DATA EXTRACTION AND SYNTHESIS: The characteristics of the included studies-age, sex, type of study, sample size, caries at baseline, setting, operator, blinding, intervention, outcomes and assessment of any confounders-were extracted from the included papers. The quality assessment was carried out using the Cochrane risk of bias tool. The success rate and odds ratios were chosen to calculate the effect size for the meta-analysis. RESULTS: A total of nine publications were included for qualitative review and five of them were included in the meta-analysis. Around half of lesions that received annual or biannual application SDF ≥ 38% were arrested. CONCLUSIONS: SDF 38% application was found to be effective in arresting dental caries progression in cavitated primary molars.

Reversible electrical switching of spin polarization in multiferroic tunnel junctions.

Spin-polarized transport in ferromagnetic tunnel junctions, characterized by tunnel magnetoresistance, has already been proven to have great potential for application in the field of spintronics and in magnetic random access memories. Until recently, in such a junction the insulating barrier played only a passive role, namely to facilitate electron tunnelling between the ferromagnetic electrodes. However, new possibilities emerged when ferroelectric materials were used for the insulating barrier, as these possess a permanent dielectric polarization switchable between two stable states. Adding to the two different magnetization alignments of the electrode, four non-volatile states are therefore possible in such multiferroic tunnel junctions. Here, we show that owing to the coupling between magnetization and ferroelectric polarization at the interface between the electrode and barrier of a multiferroic tunnel junction, the spin polarization of the tunnelling electrons can be reversibly and remanently inverted by switching the ferroelectric polarization of the barrier. Selecting the spin direction of the tunnelling electrons by short electric pulses in the nanosecond range rather than by an applied magnetic field enables new possibilities for spin control in spintronic devices.

Dendritic cell, monocyte and T cell activation and response to glatiramer acetate in multiple sclerosis.

BACKGROUND: Treatment with glatiramer acetate (GA) modestly decreases disease activity in multiple sclerosis (MS). The mechanism of action is incompletely understood and differences in the response to treatment between individuals may exist. OBJECTIVE: To study the activation of CD4+ T cells, monocytes and dendritic cells (DC) in relation to disease activity in MS patients treated with GA. METHODS: Flow cytometry was used to study the activation of CD4+ T cells and T cell subsets (CD25(high) and CD26(high) cells), monocytes and DCs in a cross-sectional study of 39 untreated and 29 GA-treated MS patients, the latter followed prospectively for one year. Gd-enhanced magnetic resonance imaging (MRI) studies were conducted in all patients. Disease activity was assessed as relapses. RESULTS: The median percentage of DCs expressing CD40 was 10% in untreated MS patients and 5.9% in GA-treated patients (Bonferroni-corrected p=0.0005). The hazard ratio of relapse was 1.32 (95% confidence interval 1.05-1.64) per 1% increase in CD40+ DCs. Patients treated with GA had fewer CD4+ T cells expressing surface markers associated with T helper type 1 effector responses and more CD4+ T cells expressing surface markers associated with regulatory, naïve or central memory T cell populations, but CD4+ T cell activation was not related with relapse risk. CONCLUSIONS: MS patients treated with GA show prominent changes in circulating antigen-presenting cells and CD4+ T cells. Expression of CD40 on DCs is significantly lower and associated with relapse risk in MS patients treated with GA.

Stabilization of ferromagnetic order in La(0.7)Sr(0.3)MnO3-SrRuO3 Superlattices.

The study of spatially confined complex oxides is of wide interest, since correlated electrons at interfaces might form exotic phases. Here La(0.7)Sr(0.3)MnO(3)/SrRuO(3) superlattices with coherently grown interfaces were studied by structural techniques, magnetization, and magnetotransport measurements. Magnetization measurements showed that ferromagnetic order in ultrathin La(0.7)Sr(0.3)MnO(3) layers is stabilized in the superlattices down to layer thicknesses of at least two unit cells. This stabilization is destroyed, if the ferromagnetic layers are separated by two unit cell thick SrTiO(3) layers. The resistivity of the superlattices showed metallic behavior and was dominated by the conducting SrRuO(3) layers, the off-diagonal resistivity showed an anomalous Hall effect from both SrRuO(3) and La(0.7)Sr(0.3)MnO(3) layers. This shows that the La(0.7)Sr(0.3)MnO(3) layers are not only ferromagnetic but also highly conducting; probably a conducting hole gas is induced at the interfaces that stabilizes the ferromagnetic order. This result opens up an alternative route for the fabrication of two-dimensional systems with long-range ferromagnetic order.

Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis.

BACKGROUND: Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent. OBJECTIVES: We studied the immunological response to GA and its relationship with disease activity. METHODS: Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing-remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI. RESULTS: The expression of T helper type 1 (Th1) and Th17 cytokines and transcription factors was reduced during long-term treatment, but there was no relationship between the expression of cytokines and transcription factors and anti-GA antibodies. High expression of mRNA encoding GATA3 and lymphotoxin-ß (LT-ß) was associated with low disease activity in Gd-enhanced MRI studies. None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment. CONCLUSIONS: The observed relationship between the expression of mRNA encoding GATA3 and LT-ß expression and MRI disease activity deserves further analysis in future studies. The development of anti-GA antibodies was observed in all patients treated with GA, but this was not related with measures of cellular immunity, clinical or MRI disease activity.

Pathways between Social Capital and Oral Health from Childhood to Adolescence.

This study aimed to evaluate the theoretical pathways by which social capital can influence dental caries and oral health-related quality of life (OHRQoL) of children over time. This 10-y prospective cohort started in 2010 with a sample of 639 preschoolers aged 1 to 5 y from the southern Brazil. Community and individual social capital were assessed at baseline through the presence of formal institutions in the neighborhood and social networks, respectively. In the 10-y follow-up, the individual social capital was evaluated by social trust and social networks. Dental caries was measured by the International Caries Detection and Assessment System (ICDAS), and the short version of the Child Perception Questionnaire (CPQ11-14) was used to assess OHRQoL. Demographic, socioeconomic, behavioral (frequency of toothbrushing and use of dental services), and psychosocial (sense of coherence) characteristics were also assessed. Structural equation modeling was used to evaluate the associations between variables over time. About 429 children were reassessed at 10-y follow-up (67.1% cohort retention rate). High community social capital at baseline directly predicted lower occurrence of dental caries and better OHRQoL after 10 y. Social capital at community level also indirectly predicted lower occurrence of dental caries through sense of coherence, frequency of toothbrushing, and use of dental services. Individual social capital at follow-up was indirectly linked to OHRQoL via the psychosocial pathway (sense of coherence). Community-level social capital was associated with dental caries and OHRQoL over time. The relationship between individual social capital and oral health was mediated through the psychosocial pathway.

Disease protection and interleukin-10 induction by endogenous interferon-ß in multiple sclerosis?

OBJECTIVE: An immune activation response resembling virus or type I interferon responses has been observed in untreated multiple sclerosis (MS), but its pathogenic significance is uncertain. We studied the relationship between a type I interferon-like response in untreated patients with MS and disease activity. METHODS: Gene expression was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR) in whole blood samples and by microarray analysis of mononuclear cells from untreated patients with MS, patients with MS treated with IFN-ß, and patients with MS with anti-IFN-ß neutralizing antibodies (NAb). Disease activity was assessed by gadolinium-enhanced magnetic resonance imaging. RESULTS: Eight of 36 untreated patients with MS had spontaneously increased expression of the type I IFN-induced gene MX1. Microarray gene expression analysis demonstrated that patients with increased spontaneous MX1 expression also had increased expression of other genes induced by regular IFN-ß treatment of MS. MX1 expression correlated with FOXP3 and IL10 expression, and IL10 expression correlated negatively with disease activity on magnetic resonance imaging. Further, in vivo IL10 expression was lower in NAb-positive patients than in untreated patients with MS and healthy controls. Finally, ex vivo treatment of mononuclear blood cells with IFN-ß induced the expression of IL10, and this was blocked by the addition of serum from NAb-positive patients with MS. CONCLUSION: Our findings suggest that endogenous IFN-ß may induce the expression of immunoregulatory IL10 in MS and that this might be associated with dampening of inflammatory disease activity.

A retrospective clinical study on the resin infiltration of proximal caries lesions: the operator's effect.

PURPOSE: This retrospective university-based study investigated the effect of operators' training and previous experience on the success of resin infiltration (RI) in arresting proximal non-cavitated caries lesions in primary and permanent teeth. METHODS: Information was collected regarding RI of proximal non-cavitated caries lesions in primary and permanent teeth with a follow-up period up to 32 months. Factors investigated were: operators' clinical experience and training, patient's age, tooth, arch, mouth-side, surface treated, tooth separation, and baseline lesion depth. Kaplan-Meier survival and Cox regression analysis with shared frailty were used (α = 5%). RESULTS: A total of 130 proximal surfaces treated on 115 teeth of 43 children (11 ± 4.4 years) were evaluated. Survival of RI was 46% up to 32 months. Lesions treated by non-trained dentists were more likely-to-present progression than those performed by non-trained dental students under supervision (HR 2.41, 95% CI: 1.00-5.80); conversely, no difference was found between non-trained dental students under supervision and trained dentists (HR 0.52, 95% CI: 0.16-1.70). Additionally, dentin lesions were 59% more-likely-to-present progression than enamel lesions (HR 0.41, 95% CI: 0.17-0.99). CONCLUSION: The operator's experience and training could influence the success of RI on proximal non-cavitated caries lesions and it should be taken into consideration when choosing this treatment modality.

Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia.

Cytokines secreted in response to invading micro-organisms are important mediators of detrimental hemodynamic and metabolic changes in the host. To test whether cachectin/TNF plays a role in triggering release of other cytokines in the setting of infection, anesthetized baboons were passively immunized against systemic cachectin/TNF before infusion of a LD100 dose of live Escherichia coli. Bacteremia led to significant increases in circulating levels of cachectin/TNF, IL-1 beta, and IL-6. Although bacterial endotoxin/lipopolysaccharide is a potent stimulus for the synthesis and release of IL-1 and IL-6 in vitro, specific neutralization of cachectin/TNF in vivo with mAb pretreatment significantly attenuated both the IL-1 beta and the IL-6 responses despite fulminant overwhelming bacteremia. These data suggest that cachectin/TNF is essential for the initiation or amplification of IL-1 and IL-6 release during lethal gram-negative septic shock syndrome.

Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation.

Cachexia is a potentially lethal syndrome of unknown etiology characterized by anorexia, weight loss, and protein wasting that frequently complicates the treatment of chronic inflammation and cancer. Cachectin/TNF was isolated during the search for a humoral mediator of cachexia and found to stimulate the breakdown of energy stores from adipocytes and myocytes in vitro, but the chronic effects of the monokine in vivo are not known. Sublethal doses of recombinant human cachectin administered twice daily for 7-10 d caused cachexia in rats, as evidenced by reduced food intake, weight loss, and depletion of whole-body lipid and protein stores. Significant anemia is also observed and found to be the result of decreased red blood cell mass, not expanded plasma volume. Leukocytosis and histopathological evidence of tissue injury and inflammation are observed in several organs, including omentum, liver, spleen, and heart. These data suggests that the exposure of the normal host to cachectin is capable of inducing a pathophysiological syndrome of cachexia, anemia, and inflammation similar to that observed during inflammatory states or malignancy.

Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.

We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.

Tailoring magnetic interlayer coupling in La0.7Sr0.3MnO3/SrRuO3 superlattices.

The magnetic interlayer coupling in La0.7Sr0.3MnO3/SrRuO3 superlattices was investigated. High quality superlattices with ultrathin La0.7Sr0.3MnO3 and SrRuO3 layers were fabricated by pulsed laser deposition. The superlattices grew coherently with Mn/Ru intermixing restricted to about one interfacial monolayer. Strong antiferromagnetic interlayer coupling depended delicately on magnetocrystalline anisotropy and intermixing at interfaces. Ab initio calculations elucidated that the antiferromagnetic coupling is mediated by the Mn-O-Ru bond. The theoretical calculations allowed for a quantitative correlation between the total magnetic moment of the superlattice and the degree of Mn/Ru intermixing.

Methylprednisolone does not restore biological response in multiple sclerosis patients with neutralizing antibodies against interferon-ß.

BACKGROUND AND PURPOSE: Neutralizing antibodies (NAbs) appearing during treatment with Interferon-beta (IFN-beta) reduce or abolish bioactivity and therapeutic efficacy. Initial combination therapy with methylprednisolone (MP) may reduce the frequency of NAb positive patients. We hypothesized that MP treatment might also reduce NAb levels and re-establish IFN-beta bioactivity in patients already NAb+, who discontinue IFN-beta therapy. METHODS: In a 6-month open-label trial, we compared monthly high-dose pulsed MP treatment in 38 Nab positive patients with 35 NAb+, MP-untreated control patients discontinuing any therapy or switching to glatiramer acetate. All patients were NAb+ with an absent in vivo response to IFN-beta. NAbs were measured using a cytopathic effect assay and expressed as neutralizing capacity (NC) in percentage of added IFN-beta. Bioactivity was expressed as in vivo Myxovirus Resistance Protein A (MxA) mRNA induction in whole blood using real time PCR. RESULTS: At the end of study, median NAb NC was 92% in both groups. Eight patients (21%) in the MP group and four patients (11%) in the control group had regained an in vivo MxA response to IFN-beta (P = 0.35). CONCLUSIONS: Monthly pulsed MP treatment in NAb positive patients has no beneficial effect on NAb status or IFN-beta bioactivity.

Identification of new sensitive biomarkers for the in vivo response to interferon-beta treatment in multiple sclerosis using DNA-array evaluation.

OBJECTIVE: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)-beta. NAbs impair the effect of treatment. The biological effect of IFN-beta can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN-beta. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN-beta, and measured their expression in MS patients with different NAb levels. METHODS: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9-12 h and 36-48 h after IFN-beta administration. The expression of selected genes was measured by real-time PCR. NAb levels were assessed by a cytopathic effect assay. RESULTS: Several hundred genes were induced 9-12 h after an injection of IFN-beta. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36-48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real-time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. CONCLUSION: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN-beta. The expression of these markers adequately reflects bioactivity of IFN-ss as documented by the decreased induction in low NAb-positive patients and the lost induction in patients with moderate/high NAb levels.

Emergency department-initiated tobacco control: a randomised controlled trial in an inner city university hospital.

OBJECTIVES: Emergency department (ED) patients show high smoking rates. The effects of ED-initiated tobacco control (ETC) on 7-day abstinence at 12 months were investigated. METHODS: A randomised controlled intention-to-treat trial (trials registry no.: ISRCTN41527831) was conducted with 1044 patients in an urban ED. ETC consisted of on-site counselling plus up to four telephone booster sessions. Controls received usual care. Analysis was by logistic regression. RESULTS: In all, 630 (60.7%) participants were males, the median age was 30 years (range 18-81) and the median smoking intensity was 15 (range 1-60) cigarettes per day. Overall, 580 study participants (55.6%) were unmotivated, 331 (31.7%) were ambivalent and 133 (12.7%) were motivated smokers. ETC (median time 30 (range 1-99) min) was administered to 472 (91.7% out of 515) randomised study participants. At follow-up, 685 study participants (65.6% of 1044) could be contacted. In the ETC group, 73 out of 515 (14.2%) in the ETC group were abstinent, whereas 60 out of 529 (11.3%) controls were abstinent (OR adjusted for age and gender = 1.31 (95% CI 0.91 to 1.89, p = 0.15). Stratified for motivation to change behaviour, the adjusted ORs for ETC versus usual care were OR = 1.00 (95% CI 0.57 to 1.76) in unmotivated smokers, respectively OR = 1.37 (95% CI 0.73 to 2.58) in ambivalent smokers and OR = 2.19 (95% CI 0.98 to 4.89) in motivated smokers, p for trend = 0.29. CONCLUSIONS: ETC, in the form of on-site counselling with up to four telephone booster sessions, showed no overall effect on tobacco abstinence after 12 months. A non-significant trend for a better performance of ETC in more motivated smokers was observed.

Decreased T lymphocyte migration in patients with malignancy mediated by a suppressor cell population.

The migration and concentration of lymphocytes at sites of antigenic challenge are an integral part of the expression of delayed cutaneous hypersensitivity, as well as of tumor and graft rejection. In this study, we have analyzed the migration of T lymphocytes from patients with malignancy. We used casein and concanavalin A (Con A)-stimulated mononuclear cell supernatants to stimulate T cell locomotion. Peripheral blood T lymphocytes from 30 patients with established malignancy, 10 patients with indolent malignancy or benign tumor, and 42 normal adult controls were tested. Data are expressed as a migration index (MI), which represents the difference in micrometers between the distance migrated in response to a stimulus and the distance migrated in response to media alone. We observed a marked depression in casein-stimulated T lymphocyte migration in patients with established malignancy (mean MI +/- 1 SD = 17.0 +/- 9 microns) as compared with normal adult controls (mean MI +/- 1 SD = 35.3 +/- 10 microns). Similar results were observed with migration in response to Con A supernatants. T cells from patients with established malignancy had a mean MI of 5.8 +/- 4 microns to Con A supernatants as compared with 24.5 +/- 5 for controls. This depressed migration was apparent both in the distance that cells migrated and in the number of cells that migrated into the membrane. Of 10 patients with indolent malignancy or benign tumor, T cell migration in 8 was not significantly decreased as compared with controls. When we mixed equal concentrations of normal control T lymphocytes with T lymphocytes from patients with cancer and added the mixture directly to the upper compartment of the chemotaxis chamber, the response of the normal T cells to casein was inhibited by an average of 48%. We observed inhibition of this migration of normal cells when we added as little as 10% of patient cells to normal cells. When we mixed normal control T lymphocytes from different donors and added them directly to the upper compartment of the chemotaxis chamber, T lymphocyte migration in response to casein was not significantly altered. If T cells from patients with cancer were cultured overnight, the suppressive effect on lymphocyte locomotion was lost. Our results indicate that there is a population of T lymphocytes in patients with cancer that suppress normal T lymphocyte migration. This suppressor activity may partially explain the subversion of immunosurveillance in established neoplastic states, as well as the defective inflammatory reaction to intradermal injection of antigen observed in many patients with malignancy.

Using measurements of neutralizing antibodies: the challenge of IFN-beta therapy.

Although the occurrence of neutralizing antibodies (NAbs) to interferon (IFN)-beta has been acknowledged since the pivotal trials of IFN-beta in multiple sclerosis (MS), the effect of these antibodies has for several reasons been debated. The main reason for the controversies has been insufficient knowledge of the fact that clinically relevant NAbs do not appear until 12-18 months after initiation of IFN-beta therapy which make studies of 2 years or less unsuited to assess the clinical relevance of NAbs. Further, changes in NAb affinity occur and contribute to increase NAb effects by time. The present paper reviews our current knowledge of NAbs and stresses the importance of using measurements of NAbs routinely. It is concluded that NAb titres are important for the biological response to IFN-beta. Patients with low or intermediate titres may have preserved a full or partial biological response and might still benefit from IFN-beta therapy. However, persistent high titres of NAbs indicate an abrogation of the biological response and, hence, absence of therapeutic efficacy, and this observation should lead to a change of therapy. The application of the existing information about NAbs in clinical practice would lead to improved efficacy of IFN-beta treatment for the benefit of patients with MS.

The Hall Technique 10 years on: Questions and answers.

It is ten years since the first paper on the Hall Technique was published in the British Dental Journal and almost 20 years since the technique first came to notice. Dr Norna Hall a (now retired) general dental practitioner from the north of Scotland had, for many years, been managing carious primary molar teeth by cementing preformed metal crowns over them, with no local anaesthesia, tooth preparation or carious tissue removal. This first report, a retrospective analysis of Dr Hall's treatments, caused controversy. How could simply sealing a carious lesion, with all the associated bacteria and decayed tissues, possibly be clinically successful? Since then, growing understanding that caries is essentially a biofilm driven disease rather than an infectious disease, explains why the Hall Technique, and other 'sealing in' carious lesion techniques, are successful. The intervening ten years has seen robust evidence from several randomised control trials that are either completed or underway. These have found the Hall Technique superior to comparator treatments, with success rates (no pain or infection) of 99% (UK study) and 100% (Germany) at one year, 98% and 93% over two years (UK and Germany) and 97% over five years (UK). The Hall Technique is now regarded as one of several biological management options for carious lesions in primary molars. This paper covers commonly asked questions about the Hall Technique and speculates on what lies ahead.

Activation of the fifth component of human complement, C5, without cleavage, by methionine oxidizing agents.

Purified human C5 was incubated with chloramine T (Cl-T) or N-chloro-succinimide (N-Cl-S) in barbital buffer, pH 7.2. The treatment led to C5 activation: Cl-T- and N-Cl-S-treated C5 acquired a binding site for C6; upon incubation with C6 and subsequent addition of C7, C8 and C9 a membrane attack complex formed which lysed non-sensitized guinea pig red cells (reactive lysis). While the physiological activation of C5 follows its specific cleavage, the resulting fragment C5b representing the activated C5 and expressing the C6 binding site, the treatment with the mentioned chemicals does not lead to fragmentation of the C5 protein. So, functionally, the product of the chemical treatment is C5b-like, but chemically, it comprises the whole protein; no C5a is released. Cl-T and N-Cl-S are known to more or less selectively oxidize methionine residues in proteins, dependent on the conditions. Other sensitive amino acid residues are tryptophan and cysteine. Conditions were chosen for treatment of C5 with Cl-T which exclude attack on tryptophan, and we have ensured that human C5 does not contain free cysteine residues. Further, oxidation of about 60% of the methionine residues of C5 by Cl-T was demonstrated by amino acid analysis. So, all evidence points to methionine residue(s) as the site of attack of Cl-T and probably also of N-Cl-S. The oxidation product of methionine, its sulphoxide, may cause a change in structural conformation of C5 which involves expression of the C6 binding site. Earlier it was found that oxidation of C5 by hydroxyl radicals leads to its activation without cleavage. Since the properties of this C5b-like product resemble those of the product of treatment with Cl-T and N-Cl-S, it is suggested that the formerly found activation of human C5 by hydroxyl radicals is also mediated by oxidation of methionine residue(s) in the C5 protein.

Mechanisms of complement activation by crystalline cholesterol.

The mechanism by which cholesterol crystals activate complement in human serum has been studied. Crystals treated with serum and washed with buffer contain a fixed C3/C5 convertase. Its generation is dependent on the presence of divalent cations (and of factor B). The cholesterol-fixed convertase is subject to decay and can be regenerated by factors B and D. C2 in combination with C1 is not essential but enhances the convertase formation. These findings indicate that it is predominantly the alternative C3/C5 convertase C3bBb(P) that assembles on cholesterol during exposure to human serum. By the use of different antisera and immunofluorescence a C3 fragment, probably C3b, was demonstrated on serum-treated crystals. Its fixation is resistant to washing with urea, and with buffers of differing pH: by hydroxylaminolysis the C3 fragment dissociates from the crystals. This indicates a covalent ester bond linking the labile binding site of activated C3 to the hydroxyl group of cholesterol. Cholesterol acetate does not fix C3 nor acquire a C3-cleaving activity upon contact with serum. In addition, cholesterol crystals bind factor I (C3b inactivator) and in this way may facilitate fixation and amplification of the alternative C3/C5 convertase.